Matrices exhibiting the above characteristics are obtained, according to the present invention, via the polymerization or co-polymerization of a unique class of N-mono- or di-substituted acrylamide monomers, by using methods which also belong to the present invention. Included in the present invention are also matrices obtained with mixtures of such polymers or co-polymers of the afore-mentioned acrylamides, or with mixtures of such polymers and co-polymers with agarose, dextrans or other hydrophilic polymers.
Polyacrylamide matrices, for separation in zone electrophoresis, were introduced already in 1959 by Raymond and Weintraub (Science, 130, 1959, 711-712) and further promoted for use in disc electrophoresis by Davis (Ann. N. Y. Acad. Sci. 121, 1964, 404-427), Ornstein (Ann. N. Y. Acad. Sci. 121, 1964, 321-349) and Hjerten (J. Chromatogr. 11, 1963, 66-70). Their popularity as electrophoretic supports stems from some fundamental properties, such as: a) optical transparency, including the ultraviolet; b) electrical neutrality, due to the absence of charged groups; c) possibility of synthesizing gels in a wide interval of porosities. During the years, the couple of monomers which has attained the greatest popularity has been acrylamide coupled to a cross-linker, N,N'-methylene bisacrylamide (P. G. Righetti, J. Biochem. Biophys. Methods 19, 1989, 1-20). However, several defects of such a matrix have been noticed upon prolonged use. The most dramatic drawback is its instability at alkaline pH values: after an electrophoretic run (most electrokinetic separations occur at alkaline pHs for both proteins and nucleic acids), the dangling amido bonds are partly hydrolyzed, originating carboxylic groups, which stay covalently bound to the polymer, which is thus transformed into a polyacrylate. This phenomenon generates strong electroendo-osmosis, with matrix swelling and considerable distortions. In practice, after only a single electrophoretic run, the polyacrylamide matrix cannot be re-used. This strongly limits its use in large-scale projects, such as the sequencing of the human genome, where the availability of re-usable matrices would greatly shorten the analysis time and allow for a quick progress of such a project around the world. Stable matrices would be also quite useful in capillary zone electrophoresis (CZE), where the gel cannot be extruded from the capillary when partially hydrolyzed or malfunctioning.
Another common problem is the limited range of molecular sizes which can be efficiently sieved by polyacrylamides. Such porosity range encompasses pore sizes from a few (2-3 nm) to ca. 20-30 nm in highly diluted matrices. This limits the use of polyacrylamides to protein separations, whereas agarose gels are today almost exclusively used for separation of nucleic acid fragments. Highly porous polyacrylamide matrices would thus allow fractionation also of nucleic acids in some intervals of length.
A third problem is linked to the use of the standard redox couple of catalysts: persulfate and TEMED. Since this is a redox couple, it is thus able to oxidize many substances containing amino groups (from primary to tertiary), thus producing N-oxides. Such N-oxides, which remain in the gel even after discharging excess of persulfate to the anode, are able to oxidize proteins, especially the --SH residues, to disulfide bonds (--S--S--).
Some earlier patent applications have addressed some of the problems described above and have proposed different types of monomers. In one instance (Kozulic, B. and Mosbach, K., Patent No. PCT/EP88/00515, Jun. 10, 1988) Trisacryl [N-acryloyl-tris(hydromethyl)aminomethane, NAT] has been advocated for producing hydrophilic, large pore gels for electrophoresis. The Trysacryl monomer had in fact been proposed for chromatographic support media (Girot, P. and Boschetti, E., J. Chromatogr. 213, 1981, 389-396). As it will be shown below, this monomer, while strongly hydrophilic, suffers from its inherent instability, as it degrades with zero-order kinetics. Its use for, e.g., reusable or long term storage matrices cannot be clearly advocated. In another patent application (Kozulic, B., European Patent No. 88810717.4 of Oct. 19, 1988) acrylamide-sugars have been proposed, such as N-acryloyl (or metacryloyl)-1-amino-1-deoxy-D-glucitol or the corresponding D-xylitol derivative. This class of acrylamido monomers, which certainly possess good hydrophilicity and a larger molecular mass than unsubstituted acrylamide, is also extremely unstable, as it degrades with zero-order kinetics and thus does not seem to be a valid alternative, just as poly(NAT) mentioned above. In another application (Shorr, R. and Jain, T., European Patent No. 89107791.9, Apr. 28, 1989) a broad class of N-mono- and di-substituted acrylamido monomers has been proposed as electrophoretic support media, including some of the monomers mentioned above. However, out of this vast class of potential monomers, Shorr and Jain have enucleated (and commercialized) only two preferred mixtures, as follows (verbatim quotation): "in one preferred embodiment, the polymers are formed by cross-linking polymerization of N,N-dimethylacrylamide with ethyleneglycol methacrylate. In another preferred embodiment, the polymers are formed by cross-linking polymerization of N,N-dimethylacrylamide and hydroxyethyl-methacrylate with N,N-dimethylacylamide". Also these formulations do not appear to be optimal. As it will be shown below, N,N-dimethylacylamide, and similar alkyl-substituted acrylamides, are too hydrophobic, while the various methacrylate cross-linkers are too prone to hydrolysis and hydrophobic as well. As a result of this, the commercialized product containing these formulations (Hydrolink) has to contain detergents to help in solubilizing the monomers. The corresponding emulsion often flocculates. These examples show that the problems formulated above, namely the design of new matrices possessing simultaneously a high hydrophilicity, a high resistance to hyrolysis and a larger pore size have not been addressed properly and are very far from being solved.